Homocysteine has been reported to be an independent risk factor for stroke. Scutellarin (Scu) dilates cerebral blood vessels and promotes anti-platelet aggregation; however, the mechanism by which Scu and Scu-treated exosomes protect against cerebrovascular disease is unclear. The aim of the present study was to investigate the mechanisms underlying the effects of Scu and Scu-treated exosomes on tight junction proteins in the blood-brain barrier. Rat brain microvascular endothelial cells (RBMVECs) were cultured and divided into five groups: Control, model, Scu, exosomes derived from RBMVECs and exosomes derived from RBMVECs after Scu administration. MTT, lactate dehydrogenase (LDH) and nitric oxide (NO) assays were performed to assess cell viability and injury. Reactive oxygen species (ROS) levels were detected using spectrophotometry and immunofluorescence. Western blotting and immunofluorescence were performed to measure cluster of differentiation (CD) 63, claudin 5, occludin and tight junction protein 1 (ZO1) expression. The results revealed that treatment with Scu and Scu-treated exosomes enhanced cell viability, reduced cell injury, increased NO levels, upregulated CD63, claudin 5, occludin and ZO1, and decreased LDH and ROS levels. These data suggest that Scu and Scu-treated exosomes protect homocysteine-induced RBMVECs via increased claudin 5, occludin and ZO1 expression.